1. Field of the Invention
The present invention relates to a fusion protein having an enhanced in vivo activity of an anti-pernicious anemia drug, erythropoietin (hereinafter, it is also referred to as “EPO”). More particularly, the present invention relates to a fusion protein having an enhanced EPO activity by increasing its in vivo half-life with its own amino acid sequences, that is, without increasing the glycosylation content, wherein the fusion protein contains an EPO molecule fused to a particular peptide naturally occurring in vivo.
2. Description of the Related Art
EPO is a glycoprotein having a molecular weight in the range of 30,000 to 34,000 Da, and is a hematopoietic factor promoting production and differentiation of red blood cells. The glycoprotein binds to a receptor of precursor cells of red blood cells to initiate its hematopoietic activity and causes an increase in the amounts of intracellular calcium ions, an enhancement of DNA biosynthesis and stimulation of hemoglobin formation. Also, recombinant human EPO(rhEPO) has been approved for the treatment of anemia associated with kidney failure, prematurity, hypothyroidism, malnutrition and so forth, and clinical use of rhEPO is continuously increasing. However, extensive use of rhEPO could be limited by inconvenience and high costs because rhEPO should be administered about three times a week due to its short half-life. Thus, the frequency of rhEPO administration for treatment could be reduced by maintaining an in vivo activity of EPO for a longer time.
In vivo biological activity of EPO is proportional to its in vivo half-life which has been known to be related with the content of sialic acid located at the terminus of sugar chains in EPO. Thus, the in vivo biological activity of EPO is greatly dependent upon the presence or absence of sugar chains. The types of sugar chains vary depending on cell types. Thus, when the same glycoprotein is expressed in different cells, the types of sugar chains of the protein are characteristically different depending on the cell types. It is known that bacterial cells, for example, E. coli could not attach sugar chains to its proteins. Since it is known that proteins expressed in E. coli do not have any sugar chains, EPO expressed in E. coli does not contain sugar chains. In this case, EPO is confirmed to be biologically active in vitro but not active at all in vivo. This is because EPO without sugar chains is more rapidly removed from the body, compared to EPO with sugar chains, resulting in an extremely short half-life. Consequently, the presence or absence of sugar chains in EPO plays an important role in the biological activity of EPO.
To date, a lot of researches have been vigorously carried out to increase the biological activity of EPO. Most of these researches focus on substitution of some amino acids by inducing mutation of EPO genes using mutagenesis techniques. For example, PCT/US94/09257 entitled “Erythropoietin Analogs”, filed by Amgen Inc., disclosed a method of increasing an in vivo half-life by increasing the sugar chain content in EPO through mutagenesis. Increasing an in vivo half-life through EPO dimer formation has been also attempted (A. J. Sytkowski et al., J.B.C. vol. 274, No. 35, pp 24773–24778). Other methods for increasing the in vivo biological activity of EPO include fusing a novel amino acid, peptide or protein fragment to EPO molecules using genetic engineering, and to increase the sugar chain content in EPO, specifically the amounts of sialic acids. However, the kinds of amino acids, peptides or protein fragments used in this method are very limited. In most cases, such genetic modifications may result in a decrease or loss in specific activity of protein or cause antigenicity problems frequently occurring when those substances are used in vivo.
Researches into fusion proteins or chimeric proteins, rather than EPO, have been carried out, and one of the examples thereof is a follicle stimulating hormone, which is a sex hormone (Furuhashi et al., 1995, Mol. Endocrinol). However, such proteins have not yet been applied in the field because genetically modified proteins using genetic engineering pose several problems. It is not easy to obtain a modified target protein itself, requiring highly professional skills. Also, in most cases, the activities of proteins may be undesirably decreased or removed by addition of or substitution by new amino acids.
Under the circumstances, the present inventors began extensive studies into the development of a new method of increasing the in vivo activity of EPO by fusing new amino acids, peptides or proteins to EPO molecules. In the course of carrying out these studies, it was found that a fusion protein obtained by fusing carboxy terminal peptide (hereinafter, it is also referred to as “CTP”) fragments of the β subunit of a human chorionic gonadotropin (hereinafter, it is also referred to as “HCG”) which is a protein naturally occurring in vivo, to EPO, dramatically increases the in vivo half-life of the EPO. Also, the EPO contains amino acids having the function of increasing glycosylation sites while retaining the intrinsic activity of the EPO (see Korean Patent Application No. 10-2000-0075230).